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1.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810284

RESUMO

There is an increasing interest in polyphenols, plant secondary metabolites, in terms of fruit quality and diet, mainly due to their antioxidant effect. However, the identification of key gene enzymes and their roles in the phenylpropanoid pathway in temperate fruits species remains uncertain. Apricot (Prunus armeniaca) is a Mediterranean fruit with high diversity and fruit quality properties, being an excellent source of polyphenol compounds. For a better understanding of the phenolic pathway in these fruits, we selected a set of accessions with genetic-based differences in phenolic compounds accumulation. HPLC analysis of the main phenolic compounds and transcriptional analysis of the genes involved in key steps of the polyphenol network were carried out. Phenylalanine ammonia-lyase (PAL), dihydroflavonol-4-reductase (DFR) and flavonol synthase (FLS) were the key enzymes selected. Orthologous of the genes involved in transcription of these enzymes were identified in apricot: ParPAL1, ParPAL2, ParDFR, ParFLS1 and ParFLS2. Transcriptional data of the genes involved in those critical points and their relationships with the polyphenol compounds were analyzed. Higher expression of ParDFR and ParPAL2 has been associated with red-blushed accessions. Differences in expression between paralogues could be related to the presence of a BOXCOREDCPAL cis-acting element related to the genes involved in anthocyanin synthesis ParFLS2, ParDFR and ParPAL2.


Assuntos
Metaboloma , Polifenóis/biossíntese , Prunus/metabolismo , Transcriptoma , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Fenilalanina Amônia-Liase/genética , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polifenóis/genética , Prunus/genética
2.
Sheng Wu Gong Cheng Xue Bao ; 37(2): 530-540, 2021 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-33645153

RESUMO

One-carbon compounds such as methanol and methane are cheap and readily available feedstocks for biomanufacturing. Oxidation of methanol to formaldehyde catalyzed by methanol dehydrogenase (MDH) is a key step of microbial one-carbon metabolism. A variety of MDHs that depend on different co-factors and possess different enzymatic properties have been discovered from native methylotrophs. Nicotinamide adenine dinucleotide (NAD)-dependent MDHs are widely used in constructing synthetic methylotrophs, whereas this type of MDH usually suffers from low methanol oxidation activity and low affinity to methanol. Consequently, methanol oxidation is considered as a rate-limiting step of methanol metabolism in synthetic methylotrophs. To accelerate methanol oxidation, thereby improving the methanol utilization efficiency of synthetic methylotrophs, massive researches have focused on discovery and engineering of MDHs. In this review, we summarize the ongoing efforts to discover, characterize, and engineer various types of MDHs as well as the applications of MDHs in synthetic methylotrophs. Directed evolution of MDH and construction of multi-enzyme complexes are described in detail. In the future prospective part, we discuss the potential strategies of growth-coupled protein evolution and rational protein design for acquisition of superior MDHs.


Assuntos
Oxirredutases do Álcool , Metanol , Oxirredutases do Álcool/genética , Carbono , Metano
3.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540582

RESUMO

Methanol dehydrogenase (Mdh), is a crucial enzyme for utilizing methane and methanol as carbon and energy sources in methylotrophy and synthetic methylotrophy. Engineering of Mdh, especially NAD-dependent Mdh, has thus been actively investigated to enhance methanol conversion. However, its poor catalytic activity and low methanol affinity limit its wider application. In this study, we applied a transcriptional factor-based biosensor for the direct evolution of Mdh from Lysinibacillus xylanilyticus (Lxmdh), which has a relatively high turnover rate and low KM value compared to other wild-type NAD-dependent Mdhs. A random mutant library of Lxmdh was constructed in Escherichia coli and was screened using formaldehyde-detectable biosensors by incubation with low methanol concentrations. Positive clones showing higher fluorescence were selected by fluorescence-activated cell sorting (FACS) system, and their catalytic activities toward methanol were evaluated. The successfully isolated mutants E396V, K318N, and K46E showed high activity, particularly at very low methanol concentrations. In kinetic analysis, mutant E396V, K318N, and K46E had superior methanol conversion efficiency, with 79-, 23-, and 3-fold improvements compared to the wild-type, respectively. These mutant enzymes could thus be useful for engineering synthetic methylotrophy and for enhancing methanol conversion to various useful products.


Assuntos
Oxirredutases do Álcool/genética , Bacillaceae/enzimologia , Mutação , Oxirredutases do Álcool/metabolismo , Bacillaceae/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Biossensoriais , Cinética , Metanol/metabolismo
4.
Int J Mol Sci ; 22(4)2021 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-33562355

RESUMO

Synphilin-1 has previously been identified as an interaction partner of α-Synuclein (αSyn), a primary constituent of neurodegenerative disease-linked Lewy bodies. In this study, the repercussions of a disrupted glyoxalase system and aldose reductase function on Synphilin-1 inclusion formation characteristics and cell growth were investigated. To this end, either fluorescent dsRed-tagged or non-tagged human SNCAIP, which encodes the Synphilin-1 protein, was expressed in Saccharomyces cerevisiae and Schizosaccharomyces pombe yeast strains devoid of enzymes Glo1, Glo2, and Gre3. Presented data shows that lack of Glo2 and Gre3 activity in S. cerevisiae increases the formation of large Synphilin-1 inclusions. This correlates with enhanced oxidative stress levels and an inhibitory effect on exponential growth, which is most likely caused by deregulation of autophagic degradation capacity, due to excessive Synphilin-1 aggresome build-up. These findings illustrate the detrimental impact of increased oxidation and glycation on Synphilin-1 inclusion formation. Similarly, polar-localised inclusions were observed in wild-type S. pombe cells and strains deleted for either glo1+ or glo2+. Contrary to S. cerevisiae, however, no growth defects were observed upon expression of SNCAIP. Altogether, our findings show the relevance of yeasts, especially S. cerevisiae, as complementary models to unravel mechanisms contributing to Synphilin-1 pathology in the context of neurodegenerative diseases.


Assuntos
Aldeído Redutase/antagonistas & inibidores , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Lactoilglutationa Liase/antagonistas & inibidores , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Estresse Oxidativo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Glicosilação , Humanos , Corpos de Inclusão , Lactoilglutationa Liase/genética , Lactoilglutationa Liase/metabolismo , Oxirredução , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
5.
Toxicol Lett ; 342: 50-57, 2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-33581289

RESUMO

Carbonyl reduction biotransformation pathway of anthracyclines (doxorubicin, daunorubicin) is a significant process, associated with drug metabolism and elimination. However, it also plays a pivotal role in anthracyclines-induced cardiotoxicity and cancer resistance. Herein, carbonyl reduction of eight anthracyclines, at in vivo relevant concentrations (20 µM), was studied in human liver cytosol, to describe the relationship between their structure and metabolism. Significant differences of intrinsic clearance between anthracyclines, ranging from 0,62-74,9 µL/min/mg were found and associated with data from in silico analyses, considering their binding in active sites of the main anthracyclines-reducing enzymes: carbonyl reductase 1 (CBR1) and aldo-keto reductase 1C3 (AKR1C3). Partial atomic charges of carbonyl oxygen atom were also determined and considered as a factor associated with reaction rate. Structural features, including presence or absence of side-chain hydroxy group, a configuration of sugar chain hydroxy group, and tetracyclic rings substitution, affecting anthracyclines susceptibility for carbonyl reduction were identified.


Assuntos
Aclarubicina/metabolismo , Citosol/metabolismo , Doxorrubicina/análogos & derivados , Hepatócitos/metabolismo , Oxirredutases/metabolismo , Aclarubicina/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Biotransformação , Doxorrubicina/química , Doxorrubicina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Conformação Proteica
6.
Vestn Oftalmol ; 137(1): 68-73, 2021.
Artigo em Russo | MEDLINE | ID: mdl-33610152

RESUMO

The article describes a clinical case of a 14-year old patient with RDH5 mutations (OMIM *601617) in patient with fundus albipunctatus (OMIM #136880) and characteristic biomarkers of this disease with previously described pathogenic variant of nucleotic sequence in exon 3 of the RDH5 gene (NM_002905.3:c.500G>A), causing a missense change (p.Arg167His) in heterozygous state and previously not described pathogenic variant of nucleotic sequence in exon 5 of the RDH5 gene (NM_002905.3:c.838C>T), leading to a missense change (p.Arg280Cys) in heterozygous state with characteristic biomarkers of the disease. Best-corrected visual acuity (BCVA) was 20/20. Nyctalopia was accompanied by reduced b-wave of scotopic (dark-adapted 0.01) ERG and decreased amplitude of a- and b-waves of maximum (dark-adapted 3) ERG. Decreased amplitude of the a- and b-waves of photopic (light-adapted 3) ERG and the amplitude of high-frequency (light-adapted 30 Hz) Flicker ERG shows the involvement of retinal cone system in the process. Fundus autofluorescence imaging of both eyes produced fuzzy and grainy images with slight hyperfluorescence of retinal flecks. Optical coherence tomography showed focal thickening centered in the photoreceptor outer segment corresponding to the multiple discrete albipunctate dots.


Assuntos
Eletrorretinografia , Cegueira Noturna , Adolescente , Oxirredutases do Álcool/genética , Humanos , Mutação , Doenças Retinianas , Tomografia de Coerência Óptica
7.
Chemistry ; 27(20): 6283-6294, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33475219

RESUMO

Enzyme stereoselectivity control is still a major challenge. To gain insight into the molecular basis of enzyme stereo-recognition and expand the source of antiPrelog carbonyl reductase toward ß-ketoesters, rational enzyme design aiming at stereoselectivity inversion was performed. The designed variant Q139G switched the enzyme stereoselectivity toward ß-ketoesters from Prelog to antiPrelog, providing corresponding alcohols in high enantiomeric purity (89.1-99.1 % ee). More importantly, the well-known trade-off between stereoselectivity and activity was not found. Q139G exhibited higher catalytic activity than the wildtype enzyme, the enhancement of the catalytic efficiency (kcat /Km ) varied from 1.1- to 27.1-fold. Interestingly, the mutant Q139G did not lead to reversed stereoselectivity toward aromatic ketones. Analysis of enzyme-substrate complexes showed that the structural flexibility of ß-ketoesters and a newly formed cave together facilitated the formation of the antiPrelog-preferred conformation. In contrast, the relatively large and rigid structure of the aromatic ketones prevents them from forming the antiPrelog-preferred conformation.


Assuntos
Oxirredutases do Álcool , Álcoois , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Cetonas , Estereoisomerismo , Especificidade por Substrato
8.
Biochim Biophys Acta Mol Cell Res ; 1868(1): 118864, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32979423

RESUMO

Lanthanides are relative newcomers to the field of cell biology of metals; their specific incorporation into enzymes was only demonstrated in 2011, with the isolation of a bacterial lanthanide- and pyrroloquinoline quinone-dependent methanol dehydrogenase. Since that discovery, the efforts of many investigators have revealed that lanthanide utilization is widespread in environmentally important bacteria, and parallel efforts have focused on elucidating the molecular details involved in selective recognition and utilization of these metals. In this review, we discuss the particular chemical challenges and advantages associated with biology's use of lanthanides, as well as the currently known lanthano-enzymes and -proteins (the lanthanome). We also review the emerging understanding of the coordination chemistry and biology of lanthanide acquisition, trafficking, and regulatory pathways. These studies have revealed significant parallels with pathways for utilization of other metals in biology. Finally, we discuss some of the many unresolved questions in this burgeoning field and their potentially far-reaching applications.


Assuntos
Oxirredutases do Álcool/genética , Bactérias/genética , Elementos da Série dos Lantanídeos/metabolismo , Transporte Proteico/genética , Oxirredutases do Álcool/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos da Série dos Lantanídeos/química , Metais/metabolismo
9.
Biochim Biophys Acta Mol Basis Dis ; 1867(1): 165981, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33002578

RESUMO

The Primary Hyperoxalurias (PH) are rare disorders of metabolism leading to excessive endogenous synthesis of oxalate and recurring calcium oxalate kidney stones. Alanine glyoxylate aminotransferase (AGT), deficient in PH type 1, is a key enzyme in limiting glyoxylate oxidation to oxalate. The affinity of AGT for its co-substrate, alanine, is low suggesting that its metabolic activity could be sub-optimal in vivo. To test this hypothesis, we examined the effect of L-alanine supplementation on oxalate synthesis in cell culture and in mouse models of Primary Hyperoxaluria Type 1 (Agxt KO), Type 2 (Grhpr KO) and in wild-type mice. Our results demonstrated that increasing L-alanine in cells decreased synthesis of oxalate and increased viability of cells expressing GO and AGT when incubated with glycolate. In both wild type and Grhpr KO male and female mice, supplementation with 10% dietary L-alanine significantly decreased urinary oxalate excretion ~30% compared to baseline levels. This study demonstrates that increasing the availability of L-alanine can increase the metabolic efficiency of AGT and reduce oxalate synthesis.


Assuntos
Alanina/farmacologia , Hiperoxalúria Primária/metabolismo , Oxalatos/metabolismo , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Células CHO , Cricetulus , Hiperoxalúria Primária/genética , Hiperoxalúria Primária/patologia , Camundongos , Camundongos Knockout , Transaminases/genética , Transaminases/metabolismo
10.
PLoS One ; 15(12): e0244030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332435

RESUMO

Abiotic stresses especially salinity, drought and high temperature result in considerable reduction of crop productivity. In this study, we identified AT4G18280 annotated as a glycine-rich cell wall protein-like (hereafter refer to as GRPL1) protein as a potential multistress-responsive gene. Analysis of public transcriptome data and GUS assay of pGRPL1::GUS showed a strong induction of GRPL1 under drought, salinity and heat stresses. Transgenic plants overexpressing GRPL1-3HA showed significantly higher germination, root elongation and survival rate under salt stress. Moreover, the 35S::GRPL1-3HA transgenic lines also showed higher survival rates under drought and heat stresses. GRPL1 showed similar expression patterns with Abscisic acid (ABA)-pathway genes under different growth and stress conditions, suggesting a possibility that GRPL1 might act in the ABA pathway that is further supported by the inability of ABA-deficient mutant (aba2-1) to induce GRPL1 under drought stress. Taken together, our data presents GRPL1 as a potential multi-stress responsive gene working downstream of ABA.


Assuntos
Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Estresse Salino , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Secas , Germinação/genética , Transcriptoma
11.
Nat Commun ; 11(1): 4292, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855421

RESUMO

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.


Assuntos
Bioquímica/métodos , Butanóis/metabolismo , Enzimas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bioquímica/instrumentação , Reatores Biológicos , Sistema Livre de Células , Evolução Molecular Direcionada , Enzimas/química , Enzimas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Temperatura , Termodinâmica
12.
J Biosci Bioeng ; 130(3): 290-294, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32482608

RESUMO

Aeromonas hydrophila 4AK4 normally produces the copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) using lauric acid as the carbon source. In this study we reported the metabolic engineering of A. hydrophila 4AK4 for the production of polyhydroxyalkanoate (PHA) using acetate as a main carbon source. Recombinant A. hydrophila overexpressing ß-ketothiolase and acetoacetyl-CoA reductase could accumulate poly-3-hydroxybutyrate (PHB) from acetate with a polymer content of 1.39 wt%. Further overexpression of acetate kinase/phosphotransacetylase and acetyl-CoA synthetase improved PHB content to 8.75 wt% and 19.82 wt%, respectively. When acetate and propionate were simultaneously supplied as carbon sources, the engineered A. hydrophila overexpressing ß-ketothiolase, acetoacetyl-CoA reductase, and acetyl-CoA synthetase was found able to produce the copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV). The recombinant grew to 3.79 g/L cell dry weight (CDW) containing 15.02 wt% PHBV. Our proposed metabolic engineering strategies illustrate the feasibility for producing PHA from acetate by A. hydrophila.


Assuntos
Acetatos/metabolismo , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Engenharia Metabólica , Poli-Hidroxialcanoatos/biossíntese , Ácido 3-Hidroxibutírico/metabolismo , Acetil-CoA C-Aciltransferase/genética , Oxirredutases do Álcool/genética , Ácidos Pentanoicos/metabolismo
13.
PLoS One ; 15(5): e0232090, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32357153

RESUMO

Dihydroflavonol 4-reductase (DFR), a key enzyme involved in the biosynthesis of anthocyanins, has been cloned from various species. However, little research has been conducted on this enzyme in ferns, which occupy a unique evolutionary position. In this study, we isolated two novel DFR genes from the fern Dryopteris erythrosora. In vitro enzymatic analysis revealed that DeDFR1 and DeDFR2 enzymes can catalyze dihydrokaempferol and dihydroquercetin but cannot catalyze dihydromyricetin. Amino acid sequence analysis showed that DeDFR1 and DeDFR2 have an arginine at the same substrate-specificity-determining site as that in the ferns Salvinia cucullata and Azolla filiculoides. Thus, we speculate that the Arg-type DFR is a new DFR functional type. To further verify the substrate preferences of the Arg-type DFR, an amino acid substitution assay was conducted. When N133 was mutated to R133, Arabidopsis DFR protein completely lost its catalytic activity for dihydromyricetin, as observed for DeDFR1 and DeDFR2. Additionally, heterologous expression of DeDFR2 in the Arabidopsis tt3-1 mutant resulted in increasing anthocyanin accumulation. In summary, DeDFR1 and DeDFR2 are considered to be a new type of DFR with unique structures and functions. The discovery of the Arg-type DFR provides new insights into the anthocyanin biosynthesis pathway in ferns.


Assuntos
Oxirredutases do Álcool/genética , Antocianinas/biossíntese , Dryopteris/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Catálise , Dryopteris/enzimologia , Dryopteris/metabolismo , Mutagênese Sítio-Dirigida , Filogenia , Alinhamento de Sequência , Especificidade por Substrato
14.
Appl Environ Microbiol ; 86(13)2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32385078

RESUMO

Although Clostridium acetobutylicum is the model organism for the study of acetone-butanol-ethanol (ABE) fermentation, its characterization has long been impeded by the lack of efficient genome editing tools. In particular, the contribution of alcohol dehydrogenases to solventogenesis in this bacterium has mostly been studied with the generation of single-gene deletion strains. In this study, the three butanol dehydrogenase-encoding genes located on the chromosome of the DSM 792 reference strain were deleted iteratively by using a recently developed CRISPR-Cas9 tool improved by using an anti-CRISPR protein-encoding gene, acrIIA4 Although the literature has previously shown that inactivation of either bdhA, bdhB, or bdhC had only moderate effects on the strain, this study shows that clean deletion of both bdhA and bdhB strongly impaired solvent production and that a triple mutant ΔbdhA ΔbdhB ΔbdhC was even more affected. Complementation experiments confirmed the key role of these enzymes and the capacity of each bdh copy to fully restore efficient ABE fermentation in the triple deletion strain.IMPORTANCE An efficient CRISPR-Cas9 editing tool based on a previous two-plasmid system was developed for Clostridium acetobutylicum and used to investigate the contribution of chromosomal butanol dehydrogenase genes during solventogenesis. Thanks to the control of cas9 expression by inducible promoters and of Cas9-guide RNA (gRNA) complex activity by an anti-CRISPR protein, this genetic tool allows relatively fast, precise, markerless, and iterative modifications in the genome of this bacterium and potentially of other bacterial species. As an example, scarless mutants in which up to three genes coding for alcohol dehydrogenases are inactivated were then constructed and characterized through fermentation assays. The results obtained show that in C. acetobutylicum, other enzymes than the well-known AdhE1 are crucial for the synthesis of alcohol and, more globally, to perform efficient solventogenesis.


Assuntos
Oxirredutases do Álcool/genética , Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Clostridium acetobutylicum/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Clostridium acetobutylicum/enzimologia , Edição de Genes
15.
Sci Rep ; 10(1): 7813, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-32385385

RESUMO

The conversion of lignocellulosic biomass into bioethanol or biochemical products requires a crucial pretreatment process to breakdown the recalcitrant lignin structure. This research focuses on the isolation and characterization of a lignin-degrading bacterial strain from a decaying oil palm empty fruit bunch (OPEFB). The isolated strain, identified as Streptomyces sp. S6, grew in a minimal medium with Kraft lignin (KL) as the sole carbon source. Several known ligninolytic enzyme assays were performed, and lignin peroxidase (LiP), laccase (Lac), dye-decolorizing peroxidase (DyP) and aryl-alcohol oxidase (AAO) activities were detected. A 55.3% reduction in the molecular weight (Mw) of KL was observed after 7 days of incubation with Streptomyces sp. S6 based on gel-permeation chromatography (GPC). Gas chromatography-mass spectrometry (GC-MS) also successfully highlighted the production of lignin-derived aromatic compounds, such as 3-methyl-butanoic acid, guaiacol derivatives, and 4,6-dimethyl-dodecane, after treatment of KL with strain S6. Finally, draft genome analysis of Streptomyces sp. S6 also revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin depolymerization, as well as for the mineralization of the lower molecular weight compounds, confirming the lignin degradation capability of the bacterial strain.


Assuntos
Lignina/metabolismo , Streptomyces/enzimologia , Oxirredutases do Álcool/genética , Biodegradação Ambiental , Biomassa , Etanol/metabolismo , Lacase/genética , Lignina/química , Lignina/genética , Peroxidase/genética , Peroxidases/genética , Polimerização , Streptomyces/genética
16.
Invest Ophthalmol Vis Sci ; 61(3): 53, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232344

RESUMO

Purpose: To investigate clinical characteristics of RDH5-related fundus albipunctatus (FAP) in a Japanese cohort. Methods: Twenty-five patients from 22 pedigrees with RDH5-related FAP were studied. Ophthalmic medical records were reviewed. For genetic analysis, either Sanger sequencing of the RDH5 gene or whole-exome sequencing was performed. Results: Genetic analysis identified eight different RDH5 variants, including seven known RDH5 variants (p.G35S, p.G107R, p.R167H, p.A240GfsX19, p.R278X, p.R280H, and p.L310delinsEV) and a novel variant: c.259C>T (p.Q87X). The most frequently observed variant was p.L310delinsEV (65.2%, 30/46 alleles). Of 50 eyes examined, 44 eyes (88.0%) showed logMAR best-corrected visual acuity (BCVA) of 0.10 or better. In optical coherence tomography, macular involvement was observed in 12 patients (24 eyes). Ten patients (83.3%) who had good BCVA (0.10 or better) exhibited diffuse disruption of the outer retina with foveal sparing, and two patients (16.7%) exhibited diffuse disruption throughout the macula and decreased BCVA. Among the 24 eyes, ring-or crescent-shaped hyperautofluorescence or irregular autofluorescence around the fovea was observed in 15 eyes (83.3%) of 18 eyes examined by fundus autofluorescence imaging. Full-field electroretinography showed extinguished or severely decreased rod responses in all 23 examined patients, whereas decreased cone responses were seen in 17 patients (73.9%). Conclusions: Multimodal imaging and electroretinography of RDH5-related FAP revealed high frequencies of macular involvement in older patients and decreased cone responses. Our findings suggest that progressive macular/cone dysfunction, as well as delayed rod function, may be key phenotypic features of RDH5-related FAP.


Assuntos
Oxirredutases do Álcool/genética , Grupo com Ancestrais do Continente Asiático/genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Doenças Retinianas/genética , Doenças Retinianas/fisiopatologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Adolescente , Adulto , Idoso , Criança , Eletrorretinografia , Feminino , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Imagem Multimodal , Linhagem , Doenças Retinianas/diagnóstico por imagem , Estudos Retrospectivos , Tomografia de Coerência Óptica , Acuidade Visual/fisiologia , Sequenciamento Completo do Genoma , Adulto Jovem
17.
J Biomed Sci ; 27(1): 46, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32238162

RESUMO

BACKGROUND: Epigenetic silencing of retinoic acid (RA) signaling-related genes have been linked with the pathogenesis and clinical outcome in oral squamous cell carcinoma (OSCC) carcinogenesis. However, the precise mechanisms underlying the abnormal silencing of RA signaling-related genes in OSCC have not been well investigated. METHODS: Using combined analysis of genome-wide gene expression and methylation profile from 40 matched normal-tumor pairs of OSCC specimens, we found a set of retinoid signaling related genes are frequently hypermethylated and downregulated in OSCC patient samples, including alcohol dehydrogenase, iron containing 1 (ADHFE1) and aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), which are the important rate-limiting enzymes in synthesis of RA. The expression of ADHFE1 and ALDH1A2 in OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. The binding sites of miR-30a and miR-379 with DNA methyltransferase 3B (DNMT3B) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and Western blot analyses. The functions of miR-30a, miR-379, and DNMT3B were accessed by growth and colony formation analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) was performed to explore the molecular mechanisms by arecoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment. RESULTS: We demonstrated that deregulated miR-30a and miR-379 could represent a mechanism for the silencing of ADHFE1 and ALDH1A2 in OSCC through targeting DNMT3B. Ectopic expression of miR-30a and miR-379 could induce re-expression of methylation-silenced ADHFE1 and ALDH1A2, and lead to growth inhibition in oral cancer cells. Furthermore, the dysregulation of the miRNAs and DNMT-3B may result from exposure to tobacco smoking and betel quid chewing. CONCLUSIONS: Our results demonstrate that tobacco smoking and betel quid chewing could repress miR-30a and miR-379, which upregulate the DNMT3B expression, in turn, lead to the hypermethylation of ADHFE1 and ALDH1A genes, consequently, promote the oncogenic activity. These findings highlight the potential use of retinoids in combination with epigenetic modifiers for the prevention or treatment of oral cancer.


Assuntos
Carcinoma de Células Escamosas/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Inativação Gênica , MicroRNAs/genética , Neoplasias Bucais/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , /metabolismo , Arecolina/química , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Redes e Vias Metabólicas , MicroRNAs/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Nitrosaminas/química , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Tretinoína/metabolismo
18.
Mol Carcinog ; 59(5): 512-519, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32124501

RESUMO

Overexpression of RAD51 is found in many cancers including breast cancer and is associated with poor survival. Compared with normal cells, RAD51 promoter is hyperactive in cancer cells indicating that RAD51 is transcriptionally activated. However, little is known about the mechanisms and factors involved in RAD51 transcription regulation. Transcription corepressor, C-terminal binding protein 1 (CtBP1), is an oncogene repressing a panel of tumor suppressors transcription, which contributes to cancer progression. In this study, immunohistochemistry (IHC) revealed that RAD51 expression was positively correlated with CtBP1 expression in breast cancer patient tissues; short hairpin RNA-mediated CtBP1 depletion, chromatin immunoprecipitation, and dual-luciferase reporter assays showed that CtBP1 activated RAD51 transcription in breast cancer cells. Depletion of CtBP1 increased breast cancer cells' sensitivity to cisplatin and, in turn, expression of exogenous RAD51 in the CtBP1-depleted breast cancer cells increased resistance to cisplatin. The results demonstrated that CtBP1 conferred breast cancer cells resistance to cisplatin through transcriptional activation of RAD51.


Assuntos
Oxirredutases do Álcool/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Cisplatino/farmacologia , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Rad51 Recombinase/metabolismo , Ativação Transcricional , Oxirredutases do Álcool/genética , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Proteínas de Ligação a DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Prognóstico , Regiões Promotoras Genéticas , Rad51 Recombinase/genética , Células Tumorais Cultivadas
19.
PLoS One ; 15(3): e0230915, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32218601

RESUMO

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5' coding region and the 5' upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes , Nocardia/enzimologia , Oxirredutases do Álcool/deficiência , Biotransformação , Mutação , Nocardia/genética
20.
Microbes Environ ; 35(1)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32037377

RESUMO

Although the bioavailability of rare earth elements (REEs, including scandium, yttrium, and 15 lanthanides) has not yet been examined in detail, methane-oxidizing bacteria (methanotrophs) were recently shown to harbor specific types of methanol dehydrogenases (XoxF-MDHs) that contain lanthanides in their active site, whereas their well-characterized counterparts (MxaF-MDHs) were Ca2+-dependent. However, lanthanide dependency in methanotrophs has not been demonstrated, except in acidic environments in which the solubility of lanthanides is high. We herein report the isolation of a lanthanide-dependent methanotroph from a circumneutral environment in which lanthanides only slightly dissolved. Methanotrophs were enriched and isolated from pond sediment using mineral medium supplemented with CaCl2 or REE chlorides. A methanotroph isolated from the cerium (Ce) chloride-supplemented culture, Methylosinus sp. strain Ce-a6, was clearly dependent on lanthanide. Strain Ce-a6 only required approximately 30 nM lanthanide chloride for its optimal growth and exhibited the ability to utilize insoluble lanthanide oxides, which may enable survival in circumneutral environments. Genome and gene expression analyses revealed that strain Ce-a6 lost the ability to produce functional MxaF-MDH, and this may have been due to a large-scale deletion around the mxa gene cluster. The present results provide evidence for lanthanide dependency as a novel survival strategy by methanotrophs in circumneutral environments.


Assuntos
Genoma Bacteriano/genética , Elementos da Série dos Lantanídeos/metabolismo , Proteobactérias/genética , Proteobactérias/isolamento & purificação , Oxirredutases do Álcool/genética , Proteínas de Bactérias/genética , Meios de Cultura/metabolismo , Sedimentos Geológicos/microbiologia , Metais Terras Raras/metabolismo , Metano/metabolismo , Methylosinus/classificação , Methylosinus/genética , Methylosinus/isolamento & purificação , Methylosinus/metabolismo , Tanques/microbiologia , Proteobactérias/classificação , Proteobactérias/fisiologia , RNA Ribossômico 16S/genética
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